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Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins' structural diversity. © 2014 American Society of Plant Biologists. All rights reserved.

ANTHOCYANIN1 (AN1) of petunia is a transcription factor of the basic helix-loop-helix (bHLH) family that is required for the synthesis of anthocyanin pigments. Here, we show that AN1 controls additional aspects of cell differentiation: the acidification of vacuoles in petal cells, and the size and morphology of cells in the seed coat epidermis. We identified an1 alleles, formerly known as ph6, that sustain anthocyanin synthesis but not vacuolar acidification and seed coat morphogenesis. These alleles express truncated proteins lacking the C-terminal half of AN1, including the bHLH domain, at an ∼30-fold higher level than wild-type AN1. An allelic series in which one, two, or three amino acids were inserted into the bHLH domain indicated that this domain is required for both anthocyanin synthesis and vacuolar acidification. These findings show that AN1 controls more aspects of epidermal cell differentiation than previously thought through partially separable domains.

Fifty new flower pigmentation mutants in Petunia hybrida using endogenous transposable elements (TE(s)) as a mutagen were generated. Forty-six mutants displayed somatic and sporogenic instability indicating that they were caused by a TE. Phenotypic analysis showed that the mutation uttered either anthocyanin biosynthesis (40 alleles for seven loci), the intracellular pH of petals (six alleles for three loci) or the shape of petal cells (two alleles for two loci). To identify the TEs reponsible for the mutations the authors subjected 16 alleles of the anthocyanin-3 (an3) locus, encoding flavanone 3β-hydroxylase, to molecular for analysis. This showed that 11 out of 12 unstable an3 alleles harboured TE insertions of a single family, dTph1, while one allele harboured a new 177 bp TE designated dTph2. In addition, the authors found one an3 allele (an3-W138A) in which a dTph1 element had inserted 30 bp upstream the translation start, without inactivating the gene. This 'cryptic' element was reponsible for the creation of a stable recessive (untagged) an3 allele, where a large rearrangement inactivated the gene. These findings indicate that mutants for novel loci are most likely tagged by dTph1 elements opening the way for their isolation.